Luciferase, which is an enzyme protein capable of emitting bioluminescence through the enzymatic oxidation of luminescent substrates (luciferins), has conventionally been utilized as a reporter protein in a variety of bioassay systems. That is, genes encoding several luciferases have been collected for the purpose of being recombinantly expressed in the host cell in in-vitro cell culture systems, and recombinant expression systems thereof have also been established. Particularly, in the recombinant expression systems of luciferase, a gene encoding luciferase is transcribed under the control of a heterologous promoter in a host cell, and the transcribed mRNA is translated into a peptide chain to produce luciferase protein of interest.
The use of secreted luciferase, which is a luciferase protein recombinantly expressed in a host cell and secreted to the outside of the cell, enables the convenient monitoring of reporter protein yields by sampling a medium containing the secreted reporter protein without sampling the cultured host cell. For example, when time-dependent changes in reporter protein yield are observed in in-vitro cell culture systems, the use of the secreted luciferase enables the use of an approach comprising sampling a predetermined amount of a medium at each point in time and monitoring the concentration of secreted luciferase contained in the medium. There were previously reported secreted luciferases capable of being recombinantly expressed that can be employed in such application, such as luciferases from Cypridina (Vargula) hilgendorfii belonging to Ostracoda (Thompson, E. M., et al., Proc. Natl. Acad. Sci. USA, 86, 6567-6571 (1989)) and from Oplophorus gracilirostris belonging to Decapoda (Inouye, S., et al., FEBS Lett., 481, 19-25 (2000); JP 2002-320482 A). It have also been reported that these two types of secreted luciferases were produced by recombinant expression using animal cells as hosts to be secreted therefrom.
Furthermore, there were previously reported, as for secreted luciferases found in other origins, luciferases from Gaussia princeps (GenBank™/EBI accession number AY015993) and Metridia longa (GenBank™/EBI accession number AY364164; J. Biol. Chem. Vol. 279, No. 5 pp. 3212-3217 (2004)) both belonging to Metridinidae.
These secreted luciferases cause luminescence through the actions on their respective luminescent substrates (luciferins) specific to the organisms. For constructing a variety of bioassay systems, the luciferases are desired to be capable of acting on more versatile luminescent substrates (luciferins). For example, when the following coelenterazine is utilized as a more versatile luminescent substrate (luciferin),
Such secreted luciferase capable of causing desired luminescence is preferable for constructing a variety of bioassay systems. For example, there have been reported luciferases from Cypridina (Vargula) hilgendorfii belonging to Ostracoda, from Oplophorus gracilirostris belonging to Decapoda, from Gaussia princeps, and from Metridia longa, which are all capable of using the coelenterazine as their luminescent substrates (luciferins).